Fig 1.
Tnks DKO results in intestine degeneration and mouse lethality.
(A) Experimental outline for intestine-specific deletion of Tnks2. TAM: tamoxifen; Sac: sacrifice. (B) Immunohistological analysis of TNKSs knockout efficiency in small intestinal epithelium at day 4 after the first TAM injection. Representative images from three mice of each genotype are depicted. Scale bar: 100 μm. Ctrl and DKO indicate Tnks1+/+;Tnks2+/+ and Vil-creERT2;Tnks1-/-;Tnks2fl/fl, respectively. (C) Survival rate of control and DKO (n = 24) mice, administered TAM intraperitoneally daily for 4 times, from two different experiments. ***P < 0.001, by log rank test. (D) Left panel: Representative images of small intestines and colons from control and DKO mice at day 10 after the first TAM administration. Right panel: The statistic results of the small intestine and colon length (n = 5 mice). (E) H&E staining of small intestinal sections from the indicated adult mice at day 4 or day 5 after the first TAM administration. Left panel: Representative images from 5 mice of each genotype are depicted. Scale bar: 100 μm. Right panel: The statistics of the number of viable crypts of the indicated adult mice (n = 4). Data are represented as means ± SD, analyzed by two-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 2.
Reduced cell proliferation and increased apoptosis appear in Tnks DKO crypts.
(A, B) Immunofluorescence analysis of small intestinal (A) or colon (B) sections from mice at day 4 after the first TAM injection shows the expression of the proliferation marker Ki67. Left panel: Representative images from five mice of each genotype are depicted. Scale bar: 80 μm (A) and 100 μm (B). Right panel: Quantification of the Ki67 positive cells in crypts (n = 4 mice). (C) Small intestine crypt cells were isolated from control and Tnks DKO mice at day 4 after the first TAM injection, stained by propidium iodide (PI), and then subjected to FACS analysis. Left panel: A representative FACS result from 5 mice of each genotype is depicted. Right panel: Quantification of the percentage of dead cells (n = 6 mice). (D) TUNEL assay of small intestine of mice at day 4 after the first TAM injection. Left panel: Representative images from 4 mice of each genotype are depicted. Cell nuclei were counterstained with DAPI. Scale bar: 50 μm. Right panel: Quantification of apoptotic cells of the indicated adult mice (n = 4). 40 fields were scored for each condition. Data are represented as means ± SD, analyzed by two-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3.
Ablation of TNKSs reduces intestinal Lgr5+ stem cells.
(A) In situ hybridization analysis of intestinal stem cell marker Olfm4 expression in small intestine sections of control and Tnks DKO mice at day 4 after the first TAM injection. Left panel: Representative images from 4 mice of each genotype are depicted. Scale bar: 50 μm. Right panel: Quantification of the Olfm4 positive crypts of the indicated adult mice (n = 4). 40 fields were scored for each condition. (B) Lgr5-EGFP-expressing cells in proximal jejunum sections of Ctrl (Lgr5-EGFP-IRES-creERT2) or DKO-Lgr5 (Lgr5-EGFP-IRES-creERT2;Tnks1-/-;Tnks2fl/fl) mice at day 4 after the first TAM injection. Cell nuclei were counterstained with DAPI. Left panel: Representative images from 4 mice of each genotype are depicted. Scale bar: 50 μm. Right panel: Quantification of the GFP-positive cells of the indicated adult mice (n = 4). 40 fields were scored for each condition. (C) FACS analysis of Lgr5-EGFP-positive ISCs from small intestine crypt of the mice at day 4 after first TAM injection. Right panel: Quantification of EGFP+ cells of the indicated adult mice (n = 6). (D) Crypts of indicated mice (n = 3) at day 4 after the first TAM injection were isolated for qRT-PCR analysis of intestinal stem cell marker gene expression. (E) Organoids derived from the crypts of mice in (B) were treated by vehicle (EtOH) or 4-OHT. Left panel: Representative images were taken at 96h, from 5 independent experiments. Scale bar: 100 μm. Notes: Red arrow indicates autofluorescence in the organoid lumen. Right panel: Quantification of Lgr5-EGFP-high cells. Data from 5 independent experiments are represented as mean ± SEM, analyzed by unpaired Student’s t-test. **P<0.01. Data (A, B, C and D) are represented as means ± SD, analyzed by two-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4.
Decrease of Paneth cells and goblet cells in small intestine upon deletion of TNKSs.
(A) Histological analysis of Paneth cells by lysozyme staining in mice at day 4 and 5 after the first TAM injection. Representative images from four mice of each genotype are depicted. Notes: Red arrow indicates dislocated Paneth cells. Scale bar: 100 μm. Right panel: Quantification of Lysozyme+ cells (n = 4 mice). (B, C) Mucin 2 (B) or Chromogranin A (C) immunofluorescence staining of small intestinal sections from mice on day 5 after the first TAM injection. Cell nuclei were counterstained with DAPI. Scale bar: 100 μm. Right panel: Quantification of goblet cells (B) or enteroendocrine cells (C) from 40 fields scored for each condition (n = 4 mice). Data are represented as means ± SD, analyzed by two-way ANOVA test. **P < 0.01 and ***P < 0.001.
Fig 5.
Tnks DKO leads to decreased Wnt/β-catenin signaling activity.
(A) Crypts were isolated from the mice (n = 3) at day 3 and day 4 after the first TAM injection for qRT-PCR analysis to detect the expression of Wnt targets. Data represent mean ± SD, analyzed by two-way ANOVA test. *P<0.05 and **P<0.01. (B) Immunoblotting analysis of isolated small intestine crypts from control and DKO mice. GAPDH serves as an internal control. The representative result of three independent experiments was shown here. (C) Crypts were isolated from control and DKO mice were at day 4 after first TAM injection for immunoprecipitation (IP) with control IgG or anti-PAR antibody followed by immunoblotting with indicated antibodies. The representative result of three independent experiments was shown here. Protein expression was determined with whole-cell lysates.
Fig 6.
Activation of Wnt/β-catenin signaling by CHIR-99021 restores the growth of Tnks-deficient organoids.
(A) Small intestine organoid cultures in ENR medium with or without 4-OHT or CHIR-99021. Representative images were taken at 96h, from three independent experiments. Scale bar: 250 μm. Right panel: Quantification of organoids and organoid buds from 40 fields or 50 organoids. (B) Lgr5-EGFP+ cell analysis of small intestine organoids in ENR medium with or without 4-OHT or CHIR99021 for 96h from three independent experiments. Scale bar: 50 μm. Red arrow indicates autofluorescence of dead cells in the organoid lumen. (C) Quantification of Lgr5-EGFP+ cells from FACS analysis. (D) TUNEL assay of DKO organoids treated with 4-OHT or CHIR99021 for 96h. Apoptotic cells per organoid were accounted from 50 organoids of each condition from 3 independent experiments. (E) DKO organoids cultured for 72h in ENR medium with 4-OHT or CHIR-99021 were harvested for qRT-PCR analysis of the expression of Wnt target genes and stem cell markers. Data from three independent experiments are represented as mean ± SEM, analyzed by unpaired Student’s t-test. *P<0.05, **P<0.01 and ***P<0.001.